Prostate cancer is the most commonly diagnosed malignancy and second-most prevalent cause of cancer death in American men. The estimated death of prostate cancer in 2015 was 27,540, which accounted for about 9% of all male cancer deaths in the United States. Conventional therapies for prostate cancer include surgery, radiation, and hormone therapy. Although these treatments are relatively efficient for early stage prostate cancer, it is known that most patients with localized prostate cancer ultimately relapse. Chemotherapy is currently widely used for advanced prostate cancer treatment, but with limited success. Lack of targeted delivery, partly because of the lack of agents that possess tissue specificity, is one of the major hurdles that limit the effectiveness of cancer chemotherapy. Thus, a great deal of attention has been paid to the development of targeted drug delivery systems for prostate cancer therapy.
Prostate-specific membrane antigen (PSMA), also known as NAAG peptidase, glutamate carboxypeptidase II (GCPII), or N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), is a transmembrane glycoprotein that in humans is encoded by the FOLH1 (folate hydrolase 1) gene. Human PSMA contains 750 amino acids, including a large extracellular domain (707 amino acids), an intracellular domain (19 amino acids), and a transmembrane domain (24 amino acids). It weighs approximately 100 kDa. It is an enzyme that is a type II transmembrane glycosylated protein with folate hydrolase activity, and is known to be overexpressed not only in nearly all prostate cancer cells but also in tumor neovasculature of a variety of cancers (see: Wright Jr., G. L., et al., Urol. Oncol., 1995, 1(1):18-28; Chang, S. S., et al., Clin. Cancer Res., 1999, 5(10):2674-2681; O'Keefe, D. S., et al., Biochim. Biophys. Acta, 1998, 1443(1-2):113-127). By contrast, its expression in normal prostate epithelium tissues and other normal tissues has been reported to be 100-1000 fold lower (see: Wright Jr., G. L., et al., Urol. Oncol., 1995, 1(1):18-28; and others). Moreover, the expression level of PSMA correlates with prostate cancer progression. PSMA, therefore, is a validated target for prostate cancer therapy and has been adopted as a biomarker for diagnosis and imaging, and as a targeting receptor for prostate cancer therapy because of its overexpression in most prostate cancer cells. Aptamers and antibodies targeting PSMA have been discovered for targeted drug delivery to prostate cancer cells in the past few years (see: Barve, A., et al., J. Control. Release, 2014, 187:118-132). Although aptamers and antibodies retain high binding affinity to PSMA, their drawbacks, such as large size, possible immunogenicity and instability, may limit their applications in targeted drug delivery. In contrast, peptides are believed to have several advantages, including a small molecular weight, high permeability, great stability, less immunogenicity, ease of synthesis, and flexibility in chemical conjugation. Moreover, it has been reported that peptides can achieve high binding affinity and specificity that are comparable with antibodies (see: Pazgier, M., et al., Proc. Natl. Acad. Sci. U.S.A, 2009, 106(12):4665-4670; Huang, L., et al., J. Biol. Chem., 2003, 278(18): 15532-15540).
As a carboxyl peptidase, it has been reported that PSMA can cleave the terminal glutamate from NAAG or γ-linked polyglutamate. It was also reported that the enzymatic activity of PSMA was found elevated in prostate cancer cells, indicating its important role in prostate cancer progression by regulating angiogenesis. Therefore, it has been suggested that inhibition of the PSMA enzymatic activity could be a potential therapeutic approach for prostate cancer. Aggarwal and colleagues identified the peptide WQPDTAHHWATL (SEQ ID NO: 1), which can specifically bind to the catalytic site of PSMA and inhibit its enzymatic activity with an IC50 of 23 μM (Aggarwal, S., et al., Cancer Res., 2006, 66(18):9171-9177). However, it has been found that the GTIQPYPFSWGY (GTI) peptide (SEQ ID NO: 2) disclosed herein (see below) does not inhibit the PSMA enzymatic activity, which may be taken as indicating that the GTI peptide binds to a different site of PSMA extracellular domain (ECD) rather than the catalytic site.
Phage display has been widely used to identify peptide ligands for a wide variety of molecular targets, including proteins and various molecular moieties, cells, or animal tissues. A phage display library contains billions of different phages, and each phage retains a unique inserted peptide sequence on the surface. Phage display technology therefore provides a high-throughput tool for affinity selection. Protein-based biopanning and cell-based biopanning are the two most common strategies to identify peptide ligands, but both of them have disadvantages when they are used alone (see: Chen, Z., et al., Mol. Pharm., 2015, 12(6):2180-2188; Qin, B., et al., Pharm. Res., 2011, 28(10):2422-2434).
Binding affinity of peptide ligands to their receptor is known to be generally lower compared to antibodies. However, there are several strategies to increase the binding affinity of phage derived peptides. For example, affinity maturation is often employed to improve the binding affinity of peptide ligands by mutagenesis. After additional rounds of selection with these affinity maturation libraries, peptide ligands with higher affinity can be discovered. In addition, dimerization or tetramerization is a common approach to improve the binding affinity of peptides. For instance, it has been recently demonstrated that dimerization of an IGF2R-specific peptide improves its apparent affinity by nearly 9-fold (see: Chen, Z., et al., Mol. Pharm., 2015, 12(6):2180-2188). Modification of peptide side-chains and substitution of D-amino acids are other reported strategies to improve binding affinity. For example, Chen and colleagues replaced glycines with D-form amino acids and significantly improved the binding affinity and stability of the peptide (see: Chen, S., et al., Chembiochem, 2013, 14(11):1316-1322).
Most biopannings so far are conducted on a single target, such as a recombinant protein, a cell line, or a tissue. However, each of these methods has its own advantages and disadvantages. For example, a recombinant protein may exhibit a different conformation structure from its native form in cells. Therefore, a peptide ligand discovered by biopanning on recombinant protein may not exhibit the same affinity to its target cells in vitro and in vivo. On the other hand, the intricate and complex structures of cell membranes may lead to the discovery of a peptide ligand that binds to an unknown moiety. A recently conducted whole cell biopanning on PSMA-positive LNCaP cells identified a peptide ligand that exhibits very high affinity and specificity to LNCaP cells; however, this peptide was not PSMA-specific and its target moiety is unknown (Qin, B., et al., Pharm. Res., 2011, 28(10):2422-2434). Subsequently, future clinical application of this type of peptide ligands may be unpredictable. In addition, peptide ligands identified from in vitro biopanning may not survive the complex environment in the body after systemic administration.
In one embodiment of the invention, provided herein is a novel method for identifying PSMA-specific peptides that may be useful in vivo for prostate cancer diagnosis and therapy. In one aspect, said method includes a step of combinatorial biopanning against recombinant human PSMA extracellular domain (ECD), PSMA-positive LNCaP cells, and LNCaP xenografts in nude mice. Details of said novel method are provided below. A related embodiment of the invention herein provides novel peptides identified by using said method, which exhibit high affinity and specificity to PSMA in vitro and in vivo. One particular peptide identified is GTIQPYPFSWGY (or GTI) (SEQ ID NO: 2), which shows high affinity and specificity to PSMA in vitro and in vivo, and exhibits significantly higher uptake in tumor tissue than uptake in other tissues, including liver, kidneys, muscle, heart, lungs, and spleen. In another embodiment, it is demonstrated herein that said GTI peptide can be used in diagnosis of cancer by virtue of its ability to localize on cancer cells, such as PSMA-positive prostate cancer cells. Thus, said GTI peptide is capable of delivering attached imaging agents to the cancer cells. Illustratively, said GTI peptide was used as described herein to deliver an attached fluorescence agent to PSMA-positive prostate cancer cells. In another embodiment, it is demonstrated herein that said GTI peptide can be used in therapy of cancer cells, such as PSMA-positive prostate cancer cells, by delivering attached therapeutic cargos to the cancer cells. Illustratively, said GTI peptide was used as described herein to deliver a fused proapoptotic peptide, known to be incapable of entering the cells on its own, to PSMA-positive prostate cancer cells, resulting in cytotoxicity to the cancer cells. This demonstrates that said GTI peptide mediates internalization of the proapoptotic peptide into the cells.
Another embodiment of the invention herein provides a novel combinatorial phage biopanning procedure developed to discover PSMA-specific peptides that can potentially be used as ligands for targeted drug delivery to prostate cancer cells. This procedure includes conducting multiple rounds of biopanning against recombinant human PSMA extracellular domain (ECD), PSMA-positive LNCaP cells, and LNCaP xenografts in nude mice. In one illustrative example, five rounds of biopanning against recombinant human PSMA extracellular domain (ECD), PSMA-positive LNCaP cells, and LNCaP xenografts in nude mice were conducted, and various affinity assays were carried out to identify high-affinity peptides for PSMA ECD and PSMA-positive prostate cancer cells. Among these high affinity peptides, the GTI peptide disclosed herein shows the highest affinity as well as specificity to PSMA in prostate cancer cells. The apparent Kd values of the GTI peptide to PSMA-positive LNCaP and C4-2 cells are 8.22 μM and 8.91 μM, respectively. It is disclosed herein that the GTI peptide can specifically deliver the proapoptotic peptides to the prostate cancer cells to induce cell death. One such proapoptotic peptide, D(KLAKLAK)2, fused to the GTI peptide, is successfully delivered to LNCaP cells, inducing cell death. In a biodistribution study, the GTI peptide disclosed herein shows the highest uptake in C4-2 xenografts, while its uptake in other major organs, such as the liver and spleen, are either low or negligible. Compared to its scrambled control (random permutation of the GTI peptide), the GTI peptide exhibits higher and more specific uptake in C4-2 xenografts. All the results disclosed herein indicate that the GTI peptide is a potentially promising ligand for PSMA-targeted drug delivery for prostate cancer therapy.